Oral tolerance refers to the classic observation that oral antigen results in systemic hypo-responsiveness when an animal is challenged with the fed antigen. Oral tolerance is an active immunologic process mediated by multiple mechanisms: regulatory T cells (Tregs), anergy, and deletion depending on the dose given. Although oral antigen can suppress animal models of autoimmunity, human trials have given mixed results and a great deal remains to be learned about mechanisms underlying immune responses in the gut. My laboratory has a long-term interest in basic mechanisms of oral tolerance and its potential use as immune therapy. In the present competitive renewal we will address what we believe are critical unanswered questions in our understanding of oral tolerance. Low dose oral tolerance relies on the induction of Tregs and we have found that the aryl hydrocarbon receptor (AHR) plays an important role in the differentiation of FoxP3+ Treg and IL10+ Tr1 cells. AHR is a ligand-activated transcription factor and several ligands have been described to activate AHR. One of them, ITE, is a non-toxic endogenous mucosally associated ligand and will be a primary focus of the current application. AHR is related to oral tolerance as mice carrying a mutant AHR have a defect in the induction of oral tolerance and a defect in Tregs in the mesenteric lymph node but not spleen or thymus. Furthermore, in addition to its effect on T cells, the AHR ligand ITE affects dendritic cells (DCs). Specifically, ITE treated dendritic cells promote Treg generation and this effect is linked to retinoic acid. Since it has recently been shown that gut DCs play a crucial role in the induction of oral tolerance in retinoic dependent fashion, AHR ligands may play a role in oral tolerance by not only affecting by T cells, but by affecting dendritic cells as well. We hypothesize that AHR activation adjusts the activity of the gut associated lymphoid tissue in response to microbial and environmental cues and plays a central physiologic role in the Treg dependent pathway of oral tolerance. We will investigate the role that AHR plays in the induction of oral tolerance directly by promoting differentiation of FoxP3+ Treg and IL10+ Tr1 cells, and indirectly by interfering with the generation of effector T cells and by generating tolerogenic APCs that promote Treg differentiation. In addition, we found that AHR ligands act as immunomodulators to enhance oral tolerance. The induction of antigen-specific Tregs is viewed as a promising approach for treatment of autoimmune disorders such as multiple sclerosis and type 1 diabetes. Our investigations will provide an understanding of basic mechanisms of Treg induction in the gut which may then provide an avenue to achieve this goal. In this revised proposal we have presented new data supporting the role of AHR in gut immune responses and human immune responses, expanded our studies to include macrophages and B cells in addition to DCs, added the study of OT-II OVA TCR transgenic mice and clarified specific methodology and experimental design of experiments.